Chlamydia trachomatis (C. trachomatis) is a prokaryote. This organism includes the A, B, Ba, C, D, E, variant E, F, G, H, I, J, K, LGVI, LGVII, and LGVIII serotypes. C. trachomatis is the causative agent of trachoma (which is the greatest single cause of preventable blindness worldwide), inclusion conjunctivitis, infant pneumonitis, urethritis and lymphogranuloma venereum. Diagnosis and detection of this organism is often on the basis of pathologic or clinical findings and may be confirmed by isolation and staining techniques.
The genome of C. trachomatis includes a cryptic plasmid which is approximately 7.5 kb in size and is present in multiple copies in the organism. The presence of multiple copies makes this plasmid a good target for diagnostic assays using nucleic acid amplification techniques, such as Polymerase Chain Reaction (PCR) and Strand Displacement Amplification (SDA). Accordingly, many diagnostic companies currently manufacture DNA amplification assays that use the organism's cryptic plasmid as a target for detecting C. trachomatis in a biological sample.
However, there have been reports of C. trachomatis lacking the cryptic plasmid and such strains have been isolated from patients. Additionally, there have been reports of a variant strain of C. trachomatis, variant E, harboring a cryptic plasmid with a 377 base pair deletion, the area of which is targeted by assays used to detect C. trachomatis. Assays that target this area would therefore yield a false-negative result. Thus, new diagnostic techniques aimed at more reliably and accurately detecting C. trachomatis are desired.
A multi-plex DNA amplification reaction is a process in which several target genes are amplified and detected in a single reaction. This allows for the rapid screening and/or detection of multiple genes/organisms within a sample using a single assay. In a multi-plexing platform, a single reaction mixture is prepared with all reagents needed to amplify and detect each gene of interest. Often, however, these multiple reagents can cross-react with each other negatively affecting, or inhibiting, the amplification reacting and lead to false negative results. In addition, one particular amplification reaction within the mix of reactions may be more efficient than the other reactions and may consume certain reagents, such as nucleotides, at a faster rate limiting the availability of those reagents to the other assays in later amplification cycles. Accordingly, there is a need for rapid methods and reagents that allow for the detection of C. trachomatis that would also allow for the amplification and detection of multiple genes/organisms in a single assay format.
Another challenge associated with amplification reactions is that the DNA sample to be amplified must be extracted from the microorganism in the sample prior to amplification and detection. Current methods of isolating DNA from the organism in a sample involve multiple extraction steps that cause a delay in identification the microorganism in the biological sample. The multiple extraction steps are often required because the extraction buffers are incompatible with the amplification reaction and need to be removed from the sample prior to amplification and detection. Accordingly, there is need for rapid methods and reagents for extracting DNA from the microorganism in the sample in a manner in which the extraction reagents do not interfere with the downstream amplification reaction.